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US20040249121A1 - Process for production of cyclic peptides - Google Patents

Process for production of cyclic peptides Download PDF

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Publication number
US20040249121A1
US20040249121A1 US10/818,246 US81824604A US2004249121A1 US 20040249121 A1 US20040249121 A1 US 20040249121A1 US 81824604 A US81824604 A US 81824604A US 2004249121 A1 US2004249121 A1 US 2004249121A1
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Prior art keywords
peptide
hplc
thr
cys
protected linear
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Avi Tovi
Chaim Eidelman
Shimon Shushan
Shai Elster
Hagi Alon
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Novetide Ltd
Teva Pharmaceuticals USA Inc
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Individual
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Assigned to TEVA PHARMACEUTICALS USA, INC. reassignment TEVA PHARMACEUTICALS USA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NOVETIDE, LTD
Assigned to NOVETIDE, LTD. reassignment NOVETIDE, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ALON, HAGI, EIDELMAN, CHAIM, ELSTER, SHAI, SHUSHAN, SHIMON, TOVI, AVI
Publication of US20040249121A1 publication Critical patent/US20040249121A1/en
Priority to US12/700,373 priority patent/US20100240865A1/en
Priority to US12/700,334 priority patent/US20100137559A1/en
Assigned to NOVETIDE, LTD. reassignment NOVETIDE, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ZAOVI, GIL, BUTILCA, GABRIEL-MARCUS, IVCHENKO, ALEXANDER
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/585—Calcitonins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • C07K1/067—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups for sulfur-containing functions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/655—Somatostatins
    • C07K14/6555—Somatostatins at least 1 amino acid in D-form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/16—Oxytocins; Vasopressins; Related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid

Definitions

  • the present invention encompasses processes for the preparation and purification of cyclic peptides.
  • Somatostatin is known to possess a very broad therapeutic potential and can be administered in a wide variety of clinical applications. The mean half-life in plasma of somatostatin is extremely short, therefore reducing the potential number of possible applications of this reagent. Research was carried out with the aim of developing analogs of somatostatin which exhibited greater stability and efficacy.
  • One series of compounds which were evaluated as potentially useful somatostatin analogs were cyclic octapeptides. Evaluation of the cyclic octapeptide, octreotide, demonstrated that the compound had excellent biological activity both in vitro and in vivo (Pless J., Metabolism 41, 5-6 (1992)).
  • Octreotide has the following basic formula:
  • D-phenylalanine D-Phe
  • D-Trp D-tryptophan residue and the disulfide bridge
  • Octreotide inhibits growth hormone for a lengthy period, inhibits the secretion of glucagon to a lesser degree, and inhibits insulin secretion only in a transient manner.
  • octreotide is selective more than other somatostatin analogues in regulating the levels of growth hormone in the body and therefore, presently is indicated in acromegaly to control and reduce the plasma levels of such hormone.
  • octreotide is useful in the treatment of cellular alterations of gastroenteropancreatic endocrine origin and of certain types of tumors.
  • the synthesis of octreotide and its derivatives has been described by two general synthetic methods.
  • the first method is a solution phase procedure, based on fragment condensation, as described by Bauer et al. European Patent Application No. 29,579 (1981) and U.S. Pat. No. 4,395,403.
  • the process generally comprises removing a protecting group from a protected hexapeptide residue; linking together two peptide units by an amide bond, wherein one comprises a hexapeptide residue; converting a functional group at the N- or C-terminal end of the resulting polypeptide; and oxidizing the polypeptide.
  • the process involves a time-consuming, multi-step synthesis, and presents additional problems during the separation of octreotide from the reaction mixtures because all the synthetic steps are carried out in solution phase.
  • the second method for the synthesis of octreotide synthesizes the entire peptide chain using solid phase peptide synthesis, starting the synthesis at the threoninol residue. This method requires that the threoninol residue be protected.
  • the second synthetic process uses an aminomethyl resin upon which the threoninol residue is incorporated with the two alcohol functions protected in acetal form.
  • Mergler et al. “Peptides: Chemistry and Biology,” Proceedings of the 12 th American Peptide Symposium , Poster 292 Presentation (Smith, J. A. and Rivier J. E., Eds ESCOM, Leiden) (1991).
  • the synthesis is carried out following an Fmoc/t-Bu protection scheme; forming the disulfide bridge on a resin by oxidation of the thiol groups of the previously deprotected cysteine residues; and releasing and deprotecting the peptide with a 20% mixture of TFA/DCM.
  • Edwards et al. disclosed a solid-phase type approximation by the stepwise synthesis on a resin of the peptide D-Phe-Cys(Acm)-Phe-D-Trp(Boc)-Lys(Boc)-Thr(t-Bu)-Cys(Acm)-HMP-resin.
  • Edwards et al. J. Med. Chem . 37, 3749-3757 (1994).
  • the disulfide was prepared on the resin, and the resultant product released from the resin by means of aminolysis with threoninol.
  • the total yield reported was only 14%.
  • Arano et al. carried out another solid phase method for DTPA-octreotide.
  • Arano et al. Bioconjugate Chem ., 8, 442-446 (1997).
  • the iodine oxidation of the DTPA-peptide produced DTPA-D-Phe I -octreotide in overall 31.8% yield based on the starting Fmoc-Thr(tBu)-ol-resin.
  • Wu et al. developed a synthetic method for octreotide, wherein the disulfide bond was formed by oxidation using a dilute solution of octreotide with air during 48 hours.
  • Wu et al. Tetrahedron Letters , 39, 1783-1784 (1998).
  • Lee et al. recently carried out a new method to anchor Thr(ol) (or Thr-ol) to a solid phase synthesis resin for preparation of octreotide. See, U.S. Pat. No. 5,889,146.
  • Fmoc-Thr(ol)-terephthal-acetal was loaded onto the resin and after construction of peptide chains using Fmoc chemistry, the cyclization of the peptide was obtained on resin by oxidation with iodine.
  • the present invention encompasses processes for the preparation and purification of cyclic peptides.
  • the processes comprise (a) providing a protected linear peptide, wherein the peptide contains at least two protected thiol-containing residues, of which at least one is protected with an orthogonal protecting group; (b) reacting the protected linear peptide with an acidic composition to produce a semi-protected linear peptide protected with the orthogonal protecting group on one of the thiol-containing residues; (c) purifying the semi-protected linear peptide by chromatography; (d) treating the purified semi-protected linear peptide obtained in step (c) with an oxidizing agent to produce an unprotected cyclic peptide; and (e) purifying the unprotected cyclic peptide by chromatography.
  • the process further comprises neutralizing excess oxidizing agent after step (d).
  • the oxidizing agent may be iodine.
  • the cyclic peptide is selected from the group consisting of somatostatin analogues, vasopressin related peptides, ⁇ -atrial natriuretic factors/peptides (ANF/ANP), calcitonins and other disulfide containing peptides.
  • the cyclic peptide is octreotide, calcitonin (salmon), desmopressin, oxytocin, nesiritide, or eptifibatide.
  • the linear peptide of step (a) is attached to a resin or in solution.
  • the orthogonal protecting group is acetamidomethyl, benzyl, 4-methoxybenzyl, tert-butyl, trimethylacetamidomethyl, phenylacetamidomethyl, or tert-butylmercapto.
  • the orthogonal group is a non-acid labile protecting group such as acetamidomethyl (ACM).
  • One embodiment of the invention encompasses a cyclic peptide having a purity of at least about 98.5% by HPLC and preferably, at least about 99% by HPLC.
  • Another embodiment of the invention is the preparation of a cyclic peptide at high purity, by a process wherein the reaction product upon cleavage from a support resin, when attached to a support resin, is partially deprotected and carries at least one protecting group attached to a thiol-containing residue.
  • Another embodiment of the invention encompasses a peptide prepared from amino residues by employing Acm as a protecting group for cysteine residues.
  • the invention encompasses methods for preparing polypeptides. More specifically, the invention encompasses methods for preparing cyclic polypeptides wherein at least one protecting group attached to thiol-containing residues, such as cysteine residues, is not cleaved or deprotected from the thiol-containing residues under the conditions required for cleavage other protecting groups. Thus, the peptide chain is not completely deprotected, but carries at least one protecting group attached to a thiol-containing residue.
  • the invention encompasses processes for preparing cyclic peptides selected from the group consisting of somatostatin analogues, vasopressin related peptides, ⁇ -atrial natriuretic factors/peptides (ANF/ANP), calcitonins, and other disulfide containing peptides. More specifically, the cyclic peptide is selected from the group consisting of octreotide, calcitonin (salmon), desmopressin, oxytocin, nesiritide, and eptifibatide.
  • the reaction product may be purified to obtain a cyclic peptide of high purity.
  • high purity refers to a composition comprises at least about 98.5% as determined by HPLC, and preferably at least about 99% as determined by HPLC.
  • cyclic peptide refers to a peptide containing at least two (2) thiol-containing residues connected by a disulfide bridge.
  • the synthesis of the semi-protected peptide may be performed by known methods for peptide synthesis, for example, on a solid support or in solution, among others.
  • the process of the invention for preparing cyclic peptides is a process wherein at least one thiol-containing residue of the starting material is protected by an orthogonal protecting group that is not cleaved or deprotected from a thiol-containing residue under the conditions required for the cleavage of other protecting groups or cleavage of the peptide from the resin.
  • orthogonal protecting group refers to a protecting group that is chemically resistant under one set of selected conditions, but is liable under another set of conditions.
  • Orthogonal protecting groups include, but are not limited to, at least one of acetamidomethyl (Acm), benzyl (Bzl), 4-methoxybenzyl (Mob), tert-butyl (Tbu or t-Bu), trimethylacetamidomethyl (Tacm), phenylacetamidomethyl(Phacm), or tert-butylmercapto (StBu).
  • the orthogonal protecting group is a non-acid labile group such as acetamidomethyl.
  • the peptide chain is not completely deprotected after deprotection or cleavage, but carries at least one protecting group attached to a thiol-containing residue (a semi-protected polypeptide).
  • the semi-protected polypeptide may be purified using any suitable methods.
  • the remaining protecting groups on the semi-protected polypeptide are removed and a disulfide bridge formed to obtain a cyclic peptide using any conventional method.
  • one method includes, but is not limited to, thiol oxidation by an oxidizing agent such as iodine.
  • the cyclic peptide is purified by suitable methods to obtain a high purity cyclic peptide.
  • excess oxidizing agent can be neutralized prior to purification.
  • purification is carried out using HPLC.
  • TIS Triisopropylsilane Trt trityl Mpa mercaptopropionic acid TIS Triisopropylsilane Trt trityl Mpa mercaptopropionic acid.
  • thiol-containing residues are protected using an orthogonal protecting group such as acetamidomethyl (Acm).
  • Acm acetamidomethyl
  • acidolytic cleavage of the peptide will yield a peptide sequence carrying one Acm group.
  • octreotide in the preparation of octreotide, if Acm is used as a protecting group of a first thiol-containing residue, e.g., the cysteine residue attached to Thr-ol, acidolytic cleavage of the peptide will yield a peptide sequence carrying one Acm group, for example, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr(ol).
  • Peptides may be characterized by a specific profile of related impurities observed by HPLC analysis. Some of these impurities are easily separated from the main peak whereas others elute more closely to the main peak.
  • Coarse purification by preparative HPLC produces the partially protected peptide at a purity of at least about 95%, preferably at least about 98.5%, and most preferably, at least about 99% as determined by HPLC, and at a concentration of about 0.1 g/L to about 10 g/L.
  • Addition of an approximately equivalent amount of an oxidizing agent, such as iodine results in deprotection of the orthogonal protecting group and simultaneous cyclization of the molecule through a disulfide bridge.
  • An evaluation of the chromatographic profile at this stage shows clearly that some of the impurities that were previously eluted close to the main peak are now distinct from the product peak.
  • the product may be easily purified a second time by methods such as HPLC or other known methods to obtain a purified peptide such as octreotide in high purity.
  • HPLC high purity product is easily obtained in high yield without need for several recycling cycles that require large volumes of solvents, long operation time, and results in a lower purity and lower yield of the final product in comparison to conventional methods currently used.
  • the resulting peptide solution may be dried to obtain a dry cyclic peptide product.
  • a solid phase such as a resin and the orthogonal protecting groups are non-acid labile groups
  • the acidic composition used for deprotection of acid sensitive protecting groups also cleaves the resulting semi-protected peptide from the resin.
  • a suitable starting material may be Thr(ol)(t-Bu)-2-chlorotrityl resin.
  • step 1.2 Washing the product of step 1.1 with at least one solvent to remove all soluble compounds from the resin.
  • suitable resins for use in the process include, but are not limited to, chlorotrityl resins.
  • suitable coupling agents include, but are not limited to, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU).
  • Suitable solvents for use in the washing steps of the process include, but are not limited to, dimethylformamide (DMF), dichloromethane (DCM), methanol (MeOH), or isopropanol (IPA).
  • Suitable protecting groups for the terminal amino acid residue include, but are not limited to, 9-fluorenylmethoxycarbonyl (Fmoc) or Boc. Suitable protecting groups for the cysteine residues include Acm.
  • the terminal amino acid residue protecting group is removed by any known method, such as reaction with piperidine solution in DMF. Although one of ordinary skill in the art may substitute the reagents with other suitable reagents.
  • Cleavage of the partially protected linear intermediate from the resin support may be effected by addition of an acidic composition.
  • the acidic composition is preferably based on an acidic material such as TFA, and contains scavenger reagents including, but not limited to, ethanedithiol (EDT) and water.
  • the relative ratio of acidic material to scavenger to water may be from about 85% to about 99% acidic material, from about 0.1% to about 15% scavenger, and from about 0.1% to about 15% water by weight.
  • a preferred acidic composition comprises about 95% TFA, about 2.5% EDT, and about 2.5% water.
  • the crude peptide product may be purified by any known method.
  • the peptide is purified using HPLC on a reverse phase (RP) column.
  • RP reverse phase
  • the resulting purified product is dried and may be lyophilized.
  • Particular embodiments of the process of the present invention include, but are not limited to, the synthesis of octreotide, eptifibatide, desmopressin, and calcitonin salmon, as exemplified below.
  • One embodiment of the present invention provides a process for preparing octreotide comprising the steps of:
  • step (a) reacting the linear peptide of step (a) with an acidic composition to produce D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-ol;
  • step (b) purifying the product of step (b) by HPLC;
  • step (c) treating the resulting linear peptide of step (c) with an oxidizing agent to produce (2,7 cyclic) D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol;
  • step (d) purifying the product of step (d) by HPLC.
  • Another embodiment of the present invention provides a process for preparing eptifibatide comprising the steps of:
  • step (a) reacting the linear peptide of step (a) with an acidic composition to produce Mpa-Har-Gly-Asp-Trp-Pro-Cys(Acm)-NH 2 ;
  • step (b) purifying the product of step (b) by HPLC;
  • step (c) treating the resulting linear peptide of step (c) with an oxidizing agent to produce (1,7 cyclic) Mpa-Har-Gly-Asp-Trp-Pro-Cys(Acm)-NH 2 ;
  • step (d) purifying the product of step (d) by HPLC.
  • Yet another embodiment of the present invention provides a process for preparing desmopressin comprising the steps of:
  • step (a) reacting the linear peptide of step (a) with an acidic composition to produce Mpa-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-D-Arg-Gly-NH 2 ;
  • step (b) purifying the product of step (b) by HPLC;
  • step (c) treating the resulting linear peptide of step (c) with an oxidizing agent to produce (1,6 cyclic) Mpa-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-D-Arg-Gly-NH 2 ;
  • step (d) purifying the product of step (d) by HPLC.
  • Another embodiment of the present invention provides a process for preparing calcitonin (salmon) comprising the steps of:
  • step (a) reacting the linear peptide of step (a) with an acidic composition to produce Cys-Ser-Asn -Leu-Ser-Thr-Cys(Acm)-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys -Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH 2 ;
  • step (b) c) purifying the product of step (b) by HPLC;
  • step (c) treating the resulting linear peptide of step (c) with an oxidizing agent to produce (1,7 cyclic) Cys-Ser-Asn-Leu-Ser-Thr-Cys(Acm)-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu -Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH 2 ;and
  • step (d) purifying the product of step (d) by HPLC.
  • the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF for 20 min. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected except the last amino acid in the sequence, Boc-D-Phe. Trifunctional amino acids were side chain protected as follows: Thr(t-Bu), Cys(Trt), Cys(Acm), and Lys(Boc). Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the peptide-resin was washed with DMF, followed by DCM, and dried under vacuum to obtain 223 g dry peptide-resin.
  • the product was precipitated by the addition of 10 volumes of ether, filtered and dried in vacuum to obtain 111.7 g powder. It was identified by LC/MS as H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-ol.
  • H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys(Acm)-Thr-ol crude peptide 100 g, prepared as described in example (1) was purified on preparative C 18 RP-HPLC column. Fractions containing >95% pure product were combined and diluted to concentrations of about 1 g/L. An equimolar amount of iodine in acetic acid was added under vigorous mixing at room temperature and subsequently excess iodine was neutralized by small amount of ascorbic acid.
  • the resulting solution was loaded on a C 18 RP-HPLC column and purified to obtain fractions containing octreotide trifluoroacetate at a purity of >98.5%. After treatment to replace trifluoroacetate, the fractions were collected and lyophilized to obtain final dry peptide. The yield was 33 g (>98.5% pure).
  • Synthesis of the peptide was carried out by a regular stepwise Fmoc SPPS (solid phase peptide synthesis) procedure starting from 2-Cl-Trt resin (50 g).
  • the first amino acid (Fmoc-Cys(Acm)) was loaded onto the resin in a preliminary step to provide loading of about 0.7 mmol/g.
  • a second amino acid (Fmoc-Pro) was introduced to start the first coupling step.
  • Fmoc protected amino acid was activated in situ using TBTU/HOBt and subsequently coupled to the resin for 50 minutes. Diisopropylethylamine or Collidine were used during coupling as an organic base. Completion of the coupling was indicated by ninhydrine test.
  • the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF for 20 min. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected except the last building block in the sequence, Trt-Mpa. Trifunctional amino acids were side chain protected as follows: Asp(tBu), Har(Pbf), and Cys(Acm). Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the peptide-resin was washed with DMF, followed by DCM, and dried under vacuum to obtain 80 g dry peptide-resin.
  • the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF for 20 min. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected except the last building block in the sequence, Trt-Mpa. Trifunctional amino acids were side chain protected as follows: Gln(Trt), D-Arg(Pbf), Tyr(tBu) and Cys(Acm). Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the peptide-resin was washed with DMF, followed by DCM, and dried under vacuum to obtain 91 g dry peptide-resin.
  • the product was precipitated by the addition of 10 volumes of ether, filtered and dried in vacuum to obtain 49.0 g powder. It was identified by LC/MS as Mpa-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-D-Arg-Gly-NH 2 .
  • Synthesis of the peptide is carried out by a regular stepwise “solution synthesis” method.
  • the second amino acid (Fmoc-D-Arg(Pbf)-OH) is dissolved in DMF and pre-activated by addition of TBTU/HOBt in the presence of DIPEA.
  • the first amino acid (Gly-NH 2 ) is dissolved in DMF, is added, and the reaction continues for about 1 h at room temperature. DMF is removed under low pressure and the residue is dissolved in ethylacetate.
  • the organic solution is washed several times with aqueous HCl (1N), water and, NaHCO 3 (5%).
  • Fmoc-D-Arg(Pbf)-Gly-NH 2 Fmoc group is removed by dissolution in piperidine/DMF (20%). The solution is concentrated and the crude di-peptide is precipitated in cold ether. By a similar procedure the rest of amino acids are added sequentially to obtain final protected linear peptide. Fmoc protected amino acids are activated in situ using TBTU/HOBt and subsequently coupled to the growing peptide chain. Diisopropylethylamine or collidine are used during coupling as an organic base. Completion of the coupling is determined by HPLC or TLC test. These steps are repeated each time with another amino acid according to the peptide sequence.
  • the peptide, prepared as described above, is deprotected from its acid-labile protecting groups using a 91.5% TFA, 1.0% TIS, 2.5% EDT, 5.0% water solution for 1.5 hours at room temperature.
  • the crude product, Mpa-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-D-Arg-Gly-NH 2 is precipitated by the addition of 10 volumes of ether, filtered, and dried in a vacuum to obtain fine powder.
  • the product is identified by LC/MS.
  • the Fmoc protecting group on the ⁇ -amine was removed with 20% piperidine in DMF for 20 min. These steps were repeated each time with another amino acid according to peptide sequence. All amino acids used were Fmoc-N ⁇ protected. Trifunctional amino acids were side chain protected as follows: Cys(Trt), Ser(tBu), Asn(Trt), Gln(Trt), Thr(tBu), Glu(tBu), His(Trt), Lys(Boc), Arg(Pbf), Tyr(tBu) and Cys(Acm). Three equivalents of the activated amino acids were employed in the coupling reactions. At the end of the synthesis the peptide-resin was washed with DMF, followed by DCM, and dried under vacuum to obtain 77 g dry peptide-resin.

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US10/818,246 2003-04-07 2004-04-05 Process for production of cyclic peptides Abandoned US20040249121A1 (en)

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Application Number Priority Date Filing Date Title
US10/818,246 US20040249121A1 (en) 2003-04-07 2004-04-05 Process for production of cyclic peptides
US12/700,373 US20100240865A1 (en) 2003-04-07 2010-02-04 Process for production of cyclic peptides
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US20060148699A1 (en) * 2004-10-04 2006-07-06 Avi Tovi Counterion exchange process for peptides
US20070249806A1 (en) * 2004-10-10 2007-10-25 Saksena Divya L Solid phase Fmoc chemistry process to prepare peptides
WO2009150657A1 (en) * 2008-06-09 2009-12-17 Natco Pharma Limited Improved process for preparation of eptifibatide by fmoc solid phase synthesis
WO2010119450A2 (en) 2009-04-06 2010-10-21 Matrix Laboratories Ltd An improved process for the preparation of desmopressin or its pharmaceutically acceptable salts
WO2011156686A2 (en) * 2010-06-11 2011-12-15 The Regents Of The University Of Colorado, A Body Corporate Method for synthesizing a cyclic multivalent peptide using a thiol-mediated reaction
US8377891B2 (en) 2008-11-07 2013-02-19 Usv, Ltd. Process for synthesis of cyclic octapeptide
CN104761619A (zh) * 2015-01-06 2015-07-08 苏州天马医药集团天吉生物制药有限公司 醋酸去氨加压素的固相制备工艺
US10046025B2 (en) * 2006-06-23 2018-08-14 Aegis Therapeutics, Llc Stabilizing alkylglycoside compositions and methods thereof
US10183966B2 (en) 2013-02-21 2019-01-22 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Solid phase peptide synthesis via side chain attachment
WO2021036057A1 (zh) * 2019-08-28 2021-03-04 深圳翰宇药业股份有限公司 一种固液结合合成奈西立肽的制备方法
CN114437233A (zh) * 2020-11-06 2022-05-06 湖北健翔生物制药有限公司 一种多肽二聚体的定向合成方法
CN115057913A (zh) * 2022-06-20 2022-09-16 遵义医科大学珠海校区 一种短杆菌肽s类似物gs-5的制备方法
CN116023441A (zh) * 2022-12-29 2023-04-28 江苏诺泰澳赛诺生物制药股份有限公司 一种制备纯化去氨加压素亚砜杂质的方法

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US20060036071A1 (en) * 2004-04-08 2006-02-16 Guojie Ho Processes for preparing eptifibatide
US20060148699A1 (en) * 2004-10-04 2006-07-06 Avi Tovi Counterion exchange process for peptides
US20070249806A1 (en) * 2004-10-10 2007-10-25 Saksena Divya L Solid phase Fmoc chemistry process to prepare peptides
US7897724B2 (en) * 2004-10-10 2011-03-01 Usv, Ltd. Solid phase Fmoc chemistry process to prepare peptides
US10046025B2 (en) * 2006-06-23 2018-08-14 Aegis Therapeutics, Llc Stabilizing alkylglycoside compositions and methods thereof
WO2009150657A1 (en) * 2008-06-09 2009-12-17 Natco Pharma Limited Improved process for preparation of eptifibatide by fmoc solid phase synthesis
US8377891B2 (en) 2008-11-07 2013-02-19 Usv, Ltd. Process for synthesis of cyclic octapeptide
WO2010119450A2 (en) 2009-04-06 2010-10-21 Matrix Laboratories Ltd An improved process for the preparation of desmopressin or its pharmaceutically acceptable salts
WO2011156686A3 (en) * 2010-06-11 2012-04-12 The Regents Of The University Of Colorado, A Body Corporate Method for synthesizing a cyclic multivalent peptide using a thiol-mediated reaction
WO2011156686A2 (en) * 2010-06-11 2011-12-15 The Regents Of The University Of Colorado, A Body Corporate Method for synthesizing a cyclic multivalent peptide using a thiol-mediated reaction
US10183966B2 (en) 2013-02-21 2019-01-22 Chemical & Biopharmaceutical Laboratories Of Patras S.A. Solid phase peptide synthesis via side chain attachment
CN104761619A (zh) * 2015-01-06 2015-07-08 苏州天马医药集团天吉生物制药有限公司 醋酸去氨加压素的固相制备工艺
WO2021036057A1 (zh) * 2019-08-28 2021-03-04 深圳翰宇药业股份有限公司 一种固液结合合成奈西立肽的制备方法
CN114437233A (zh) * 2020-11-06 2022-05-06 湖北健翔生物制药有限公司 一种多肽二聚体的定向合成方法
CN115057913A (zh) * 2022-06-20 2022-09-16 遵义医科大学珠海校区 一种短杆菌肽s类似物gs-5的制备方法
CN116023441A (zh) * 2022-12-29 2023-04-28 江苏诺泰澳赛诺生物制药股份有限公司 一种制备纯化去氨加压素亚砜杂质的方法

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PT1511761E (pt) 2006-11-30
US20100137559A1 (en) 2010-06-03
DE602004001727T2 (de) 2007-08-02
EP1511761A1 (en) 2005-03-09
DK1511761T3 (da) 2006-11-27
ATE334998T1 (de) 2006-08-15
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WO2004092202A1 (en) 2004-10-28
TW200505939A (en) 2005-02-16

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